Abstract
Tissue engineering strategies using microtissues as “building blocks” have high potential in regenerative medicine. Cognition of contraction dynamics involved in the in vitro self-assembly of these microtissues can be conceived as the bedrock of an effective periodontal tissue regenerative therapy. Our study was directed at evaluating the shrinkage in the rod-shaped structure of a directed self-assembly of human gingiva-derived cells (GC) and periodontal ligament-derived cells (PDLC) and developing insights into the potential mechanisms responsible for the shrinkage. GC and PDLC were seeded in non-adherent agarose molds to form rod microtissues. Cells used for the experiments were characterized using fluorescence-activated cell sorting (FACS). To assess the viability, resazurin-based cytotoxicity assays, trypan blue dye exclusion assay, MTT and live/dead staining, and histological evaluation of rods based on hematoxylin and eosin staining were performed. Rod contraction was evaluated and measured at 0, 2, 6, and 24 h and compared to L-929 cells. The role of transforming growth factor (TGF)-β signaling, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen activated protein kinase (MAPK) signaling was analyzed. Our results show that the rod microtissues were vital after 24 h. A reduction in the length of rods was seen in the 24 h period. While the recombinant TGF-β slightly reduced contraction, inhibition of TGF-β signaling did not interfere with the contraction of the rods. Interestingly, inhibition of phosphoinositide 3-kinase by LY294002 significantly delayed contraction in GC and PDLC rods. Overall, GC and PDLC have the ability to form rod microtissues which contract over time. Thus, approaches for application of these structures as “building blocks” for periodontal tissue regeneration should consider that rods have the capacity to contract substantially. Further investigation will be needed to unravel the mechanisms behind the dynamics of contraction.
Highlights
Cells have the potential to self-assemble into spheroid-shaped structures in vitro (Dean et al, 2007)
As it can be seen, cells were positively stained for mesenchymal markers CD29, CD73, CD90, TABLE 1 | Cell surface characterization of gingiva-derived cells (GC) and periodontal ligament-derived cells (PDLC) [mean ± standard deviation, in percent (%)]
The inhibitor of phosphoinositide 3-kinase (PI3K), LY294002, was witnessed to significantly increase the length and decrease the width of both gingival and periodontal rod microtissues (p < 0.05), suggesting the role of PI3K pathway in controlling the contraction dynamics of the microtissues in vitro. In this descriptive study we investigated the contraction dynamics in the rod-shaped microtissues of cells derived from gingiva and periodontal ligament
Summary
Cells have the potential to self-assemble into spheroid-shaped structures in vitro (Dean et al, 2007). Transforming growth factor beta (TGF-β), belonging to the cytokine family is involved in the regulation of cellular functions such as cell differentiation, migration, adhesion, survival, and conditioning of developmental outcome in healthy and diseased tissue (Inman et al, 2002). Both serine/threonine kinases constitute Type I and II receptor complexes involved in TGF-β signaling pathways. Our main objective was to measure the dimensional shrinkage in the rod geometry over time and apply this information to help to bridge the gap from in vitro scaffold-free functional microtissue production to their utilization in periodontal defects in vivo
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